Apoptosis is an evolutionarily conserved process essential for normal development, cellular differentiation and homeostasis of multicellular organisms. Dysregulation of apoptosis has been associated with many human diseases, including cancer, neurodegenerative disorders, autoimmune dysfunction and aging. A key step in the mechanism of apoptosis is activation of a class of cysteine proteases, called caspases, by apoptotic stimuli. Activation of caspases is regulated by adaptor proteins such as FADD and Apaf-1, which associate in a stimulus-dependent manner with the prodomains of the initiator caspases and promote their activation via oligomerization. The activity of caspases is also regulated by a balance between the amount of inhibitor of apoptosis proteins (IAPs) and proteins that disrupts IAP-caspase interaction (IAP-inhibitors), such as mammalian Smac/DIABLO and drosophila Reaper, Hid, and Grim. The overall objective of the proposed research is to gain insight into the molecular basis of the interactions between IAPs and IAP-inhibitors and the mechanism by which IAP-inhibitors activate caspases and induce apoptosis. The first specific aim will define the role of IAPs and Smac/DIABLO in the sensitivity of cells to death stimuli, through detailed biochemical, biological and gene transfer experiments. These experiments involve studying Smac/DIABLO release from the mitochondria in response to apoptotic stimuli and pro-apoptotic Bcl-2 family members, interactions of Smac with IAPs and detailed analysis of the interaction of XIAP and Smac/DIABLO with the Apaf-l-caspase-9 apoptosome. The second specific aim will functionally and biochemically characterize potential IAP inhibitors identified in this lab. Experiments are proposed to characterize the interaction of these proteins with IAPs and determine their apoptotic and caspase promoting activity in vivo and in vitro. Other experiments are proposed to identify and characterize additional novel mammalian IAP inhibitors. The third specific aim will involve designing and testing the apoptotic activity of chemically synthesized peptides derived from IAP-inhibitors. The goals of this aim are to design short cell permeable peptides based on the sequence of the IAP-binding motif of IAP-inhibitors to test the hypothesis that small cell permeable IAP-binding peptides could block IAP function by binding to an important pocket in the IAP molecule. This work is expected to yield important information on the regulation of caspase activity and apoptosis by IAPs and IAP-inhibitors. This will facilitate the development of therapeutic inhibitors of IAPs as a new anti-cancer strategy.